An IL-17-EGFR-TRAF4 axis contributes to the alleviation of lung inflammation in severe influenza

Excessive inflammation is a postulated cause of severe disease and death in respiratory virus infections. In response to severe influenza virus infection, adoptively transferred naïve hemagglutinin-specific CD4+ T cells from CD4+ TCR-transgenic 6.5 mice drive an IFN-γ-producing Th1 response in wild-type mice. It helps in virus clearance but also causes collateral damage and disease aggravation. The donor 6.5 mice have all the CD4+ T cells with TCR specificity toward influenza hemagglutinin. Still, the infected 6.5 mice do not suffer from robust inflammation and grave outcome. The initial Th1 response wanes with time, and a prominent Th17 response of recent thymic emigrants alleviates inflammation and bestows protection in 6.5 mice. Our results suggest that viral neuraminidase-activated TGF-β of the Th1 cells guides the Th17 evolution, and IL-17 signaling through the non-canonical IL-17 receptor EGFR activates the scaffold protein TRAF4 more than TRAF6 during alleviation of lung inflammation in severe influenza.


Supplementary Note 1: HA-specific CD4 + T cell response to influenza virus infection in HAspecific CD4 + TCR-transgenic 6.5 mice
All the α/β chains of CD4 + T cell receptors (TCRs) are specific for MHC Class II I-E d restricted site of hemagglutinin (HA) from A/PR/8/34 (PR8) strain influenza virus in the 6.5 TCR-transgenic mice. Upon straight infection of 2.5 ´ 10 3 plaque-forming units (p.f.u.) of PR8 influenza virus, about 25% of the HA-specific 6.5 CD4 + T cells produced archetype Th1 cytokine IFN-γ in the lungs on day 3. About 5% of the cells produced IL-17, and 1 to 2% of the cells produced both IFN-γ and IL-17 by this time. The pattern of cytokine production was altered thereafter. On day 6, less than 10% of the cells produced IFN-γ, about 25% of the cells produced IL-17, and 8 to 10% of the cells produced both IFN-γ and IL-17. The Th17 dominant response was more prominent on day 9. By this time, only 2 to 5% of the cells produced IFN-γ, more than 45% of the cells produced IL-17, and 5 to 8% of the cells produced both IFN-γ and IL-17 ( Supplementary Fig. 1). This was in association with the decline of IFN-g-producing and TNF-aproducing cells in the infected lungs during days 6 to 9 after infection ( Supplementary Fig. 2).

IL-17 and IFN-g production of lung-infiltrating HA-specific CD4 + T cells in response to influenza virus infection in HA-specific TCR-transgenic 6.5 and syngeneic wild-type mice.
HA-specific TCR-transgenic 6.5 and syngeneic wild-type mice were infected with 2.5 ´ 10 3 p.f.u. PR8 strain H1N1 influenza virus. Infected Thy 1.2 + wild-type mice received adoptive transfer of 2.5 ´ 10 6 Thy 1.1 + naïve HA-specific 6.5 CD4 + T cells at the time of infection.
Values are mean ± s.d. of IL-17 and IFN-g production by the lung-infiltrating HA-specific 6.5 CD4 + T cells on stated days after infection in at least 3 experiments, as detected with intracellular cytokine staining (n=6/group).
4 Supplementary Fig. 2 Activation profiles of lung-infiltrating HA-specific CD4 + T cells in response to influenza virus infection in HA-specific TCR-transgenic 6.5 and syngeneic wild-type mice. HAspecific TCR-transgenic 6.5 and syngeneic wild-type mice were infected with 2.5 ´ 10 3 p.f.u.
PR8 strain H1N1 influenza virus. Infected Thy 1.2 + wild-type mice received adoptive transfer of 2.5 ´ 10 6 Thy 1.1 + naïve HA-specific 6.5 CD4 + T cells at the time of infection. Expression of the stated molecules of (a) activation and (b) regulatory phenotypes of lung-infiltrating HA-specific CD4 + T cells were examined on stated days after infection, as detected with surface and intracellular cytokine staining. Values are means ± s.d. of at least three experiments (n=6/group; ***=p<0.0001; NS=Non-significant, p>0.05; two-tailed P values for unpaired t-test).

Supplementary Note 2: Decreased T-bet dominance over ROR-gt and pre-existing Th1 cellguided Th17 response.
With 0.5 ´ 10 6 naïve cells on day 0 and 1.5 ´ 10 6 naïve cells on day 4, Day 0 cells differentiated as Th1 cells with IFN-g production and Day 4 cells differentiated as Th17 cells with IL-17 production on day 8 after infection. We used Thy1.1 and Thy1.2 as markers to differentiate adoptively transferred HA-specific CD4 + donor cell batches from each other and from endogenous CD4 + T cells in recipient mice. The T-bet was dominant over ROR-gt in the the first-batch cells, and the dominance of T-bet over ROR-gt was decreased with the excessive ROR-gt activation in the second-batch cells ( Supplementary Fig. 3).

Supplementary Fig. 3
Lost T-bet dominance over ROR-gt in the Th17 response of second-batch HA-specific CD4 + T cells. Wild type mice received adoptive transfer of two batches naïve HA-specific 6.5 CD4 + T cells. A first batch of 0.5 ´ 10 6 Thy 1.1/Thy1.1 cells were transferred into syngeneic Thy 1.2/Thy 1.2 wild type mice at the time of 2.5 ´ 10 3 p.f.u. PR8 strain H1N1 influenza virus infection. The second batch of 1.5 ´ 10 6 Thy 1.1/Thy1.2 cells were transferred on day 4 after infection. Infected mice were killed on day 8 and lung cells were analyzed for stated parameters. Control mice 6 received first-or second-batch cell transfer only, with the infection. Dot-plots are representative, and other values are mean ± s.d. of at least 3 experiments (n=6/group).

Supplementary Note 3: IL-17 deficiency in the second-batch HA-specific CD4 + T cells intensifies inflammation and aggravates the disease in the two-batch adoptive transfer experiment in IL-10KO mice
Following 2.5 ´ 10 3 p.f.u. PR8 strain influenza virus infection, the inflammatory response was augmented in the two-batch adoptive transfer experiment upon use of IL-17KO instead of IL-10KO second-batch cells in infected IL-10KO recipient mice. As a result of second-batch IL-17KO instead of IL-10KO cell transfer, first-batch IL-10KO donor cells produced more IFN-g with IL-17KO than the IL-10KO second-batch cells. The IFN-g production was also more in the IL-17KO than the IL-10 KO second-batch donor cells. With IL-17 deficiency in the second-batch HA-specific CD4 + T cells, there was more virus in the lungs and more bodyweight loss in the recipient mice on Day 8 ( Supplementary Fig. 4).

Supplementary Note 4: EGFR abundance than IL-17RA in the lungs of infected mice.
We detected surface expression of EGFR and IL-17RA of lung-infiltrating CD4 + T cells after infection of 2.5 ´ 10 3 p.f.u. PR8 strain influenza virus infection in TCR transgenic 6.5 mice, wild-type mice, and IL-17KO mice. We found upregulation of both the IL-17RA and EGFR on the lung-infiltrating CD4 + T cell surfaces after infection. EGFR expression was highest in the 6.5 mice, and IL-17RA was higher in wild-type and IL-17KO mice ( Supplementary Fig. 5).

EGFR and IL-17RA expressions on the surface of lung-infiltrating CD4 + T cells in infected
mice. 6.5 mice, wild-type mice and IL-17KO mice were infected with 2.5 ´ 10 3 p.f.u. PR8 strain influenza virus. Non-infected healthy mice (naïve) served as controls. Histograms are representatives.